Bio-Manufacturing Process

ABSTRACT

The process of making a biocomposite material utilize a bacterial species and a fungal species in an agricultural feedstock composed of a substrate of non-nutrient discrete particles and a nutrient material wherein the bacterial species imparts mechanical properties to the biocomposite material and the fungal species binds the biocomposite material. Both bacterium and fungus can be genetically engineered to produce desired properties within the microbial communities.

This is a Non-Provisional patent Application and claims the benefit of Provisional Patent Application 62/659,175, filed Mar. 28, 2018.

This invention relates to method a bio-manufacturing process. More particularly, this invention relates to method a bio-manufacturing process involving the development of a cohabitation platform incorporating reprogrammed (genetically engineered) bacterial and fungal components in order to improve existing processes of producing myceliated material.

As is known from U.S. Pat. No. 9,485,917, a composite material comprised of discrete particles and a network of interconnected mycelia cells bonding the discrete particles together can be made by inoculating a substrate of the discrete particles and a nutrient material with a preselected fungus. As described, the fungus digests the nutrient material over a period of time sufficient to grow hyphae and to allow the hyphae to form a network of interconnected mycelia cells through and around the discrete particles thereby bonding the discrete particles together to form a self-supporting composite material.

It has also been known to employ a bio-manufacturing process to make a composite material as described in U.S. Pat. No. 9,485,917 by leveraging domestic agricultural waste products, e.g. corn stalks, and inoculating these with various fungal species. The fungi utilize the agricultural substrate as the sole energy source, growing new cells (mycelia) that ramify throughout the material.

It has also been known from U.S. Pat. No. 10,125,347 to make a composite biomaterial that employs a binding organism, such as a filamentous fungi that produce mycelium, based on the material physical properties required for the composite biomaterial and a modulating organism, such as a bacteria, fungus or yeast, based on a desired effect of the modulating organism on the binding organism. As described in U.S. Pat. No. 10,125,347 a method is provided for stimulating the expression of specific tissue morphologies in filamentous fungi via interactions with competing microorganisms.

It is an object of the invention to incorporate reprogrammed (genetically engineered) bacterial and fungal components in a process of producing myceliated material.

It is another object of the invention to cohabitate both bacterial and fungal species together in a substrate of discrete particles and a nutrient material to improve existing processes of producing myceliated material and produce a new class of composite materials.

Briefly, the invention provides a process of making a biocomposite material utilizing a bacterial species and a fungal species in an agricultural feedstock composed of a substrate of non-nutrient discrete particles and a nutrient material wherein the bacterial species imparts mechanical properties to the biocomposite material and the fungal species binds the biocomposite material.

In accordance with the invention, both bacterium and fungus can be genetically engineered to produce desired properties within the microbial communities. This provides the ability to tightly regulate excreted compounds and fungal morphologies related to the production of antimicrobials, and final mechanical properties. This has also results in unique materials with a myriad of applications.

Bacterium Processing

In one embodiment, the process comprises the steps of forming a substrate of non-nutrient discrete particles and a nutrient material (i.e. a feedstock); adding a filamentous fungus to the substrate; adding a Bacillus subtilis strain characterized in producing a bio-film with poly-gamma-glutamic acid (PGA) to the substrate; and co-cultivating the fungus and the Bacillus subtilis strain in the substrate and allowing the fungus to digest the nutrient material in the substrate over a period of time sufficient to grow hyphae and to allow the hyphae to form a network of interconnected mycelia cells through and around all the non-nutrient discrete particles thereby bonding all the discrete particles together to form a self-supporting composite material.

This embodiment of the process produces a self-supporting biocomposite material comprising a substrate of non-nutrient discrete particles; a bio-film containing poly-gamma-glutamic acid (PGA) dispersed within the substrate; and a network of interconnected mycelia cells extending through and around the discrete particles and bonding the discrete particles together.

The bio-film containing poly-gamma-glutamic acid (PGA) dispersed within the substrate enhances the mechanical properties of the biocomposite material. For example, when feedstocks were co-cultivated with both fungus and PGA producing Bacillus, there was a demonstrated two-fold increase in the elastic modulus of the final material when compared to materials cultivated with only fungus.

In another embodiment, the process comprises the steps of forming a substrate of non-nutrient discrete particles and a nutrient material; adding a filamentous fungus to the substrate; adding a Bacillus subtilis strain characterized in producing melanin to the substrate; and co-cultivating the fungus and the Bacillus subtilis strain in the substrate and allowing the fungus to digest the nutrient material in the substrate over a period of time sufficient to grow hyphae and to allow the hyphae to form a network of interconnected mycelia cells through and around the non-nutrient discrete particles thereby bonding the discrete particles together to form a self-supporting composite material.

This embodiment of the process produces a self-supporting biocomposite material comprising a substrate of non-nutrient discrete particles; an amount of melanin dispersed within the substrate; and a network of interconnected mycelia cells extending through and around the discrete particles and bonding the discrete particles together.

The melanin dispersed within biocomposite material renders the biocomposite material radiation hard. Melanin is a complex molecule that is difficult to synthesize in vitro, and possesses energy absorption properties. By co-culturing a melanin producing bacteria within the composite material, one is able to manufacture melanin in situ. Once the composite is imbedded with melanin, the composite material is capable of absorbing UV and other types of radiation the material is exposed to, thus rendering the material radiation hard or resistant. The melanin imbedded material now has the capability to protect itself from dangerous radiation exposure, as well as protecting other living organisms i.e., humans, if the composite materials are used to build shelters or barriers were radiation is present i.e., laboratories or Mars.

In another embodiment, the process comprises the steps of forming a substrate of non-nutrient discrete particles and a nutrient material; adding a filamentous fungus to substrate; adding Streptomyces natalensis characterized in being able to produce natamycin during cultivation to the substrate; and co-cultivating the fungus and the Streptomyces natalensis in the substrate and allowing the fungus to digest the nutrient material in the substrate over a period of time sufficient to grow hyphae and to allow the hyphae to form a network of interconnected mycelia cells through and around the non-nutrient discrete particles thereby bonding the discrete particles together to form a self-supporting composite material while allowing the Streptomyces natalensis to produce natamycin in the self-supporting composite material.

This embodiment of the process produces a self-supporting biocomposite material comprising a substrate of non-nutrient discrete particles; an amount of natamycin dispersed within the substrate; and a network of interconnected mycelia cells extending through and around the discrete particles and bonding the discrete particles together.

The natamycin dispersed within biocomposite material imparts fungicidal properties to the biocomposite material and, in particular, renders a biocomposite material made with a filamentous fungus from the genus Ganoderma resistant to Trichoderma. Natamycin primarily targets fungi from the Ascomycota phylum. Ganoderma is from the Basidiomycota phylum.

In particular, the Streptomyces spp. is genetically engineered to produce a fungicidal agent that prevents Trichoderma spp. contamination when the material is bioactive i.e. during the manufacturing process, and throughout a living materials usable life span. This technique provides several advantages, namely, the technique

-   -   a. enables non-sterile in-field cultivation practices through         the reduction of contaminating microbes     -   b. reduces bio-control infrastructure and sterile processes         during manufacturing     -   c. provides protection against contaminants such as Trichoderma         during the usable life of “living materials”.

In another embodiment, the process comprises the steps of forming a substrate of non-nutrient discrete particles and a nutrient material; adding a filamentous fungus to the substrate; adding a Bacillus subtilis strain characterized in producing Antifungal Protein (AFP1) native to Streptomyces tendae (previously characterized by Bormann, C., Baier, D., Horr, I., Raps, C., Berger, J., Jung, G., & Schwarz, H. (1999, Sep. 27, Characterization of a Novel, Antifungal, Chitin-Binding Protein from Streptomyces tendae Tu901 That Interferes with Growth Polarity. Journal of Bacteriology, 181(24), 7421-7429) to the substrate; and co-cultivating the fungus and the Bacillus subtilis strain in the substrate and allowing the fungus to digest the nutrient material in the substrate over a period of time sufficient to grow hyphae and to allow the hyphae to form a network of interconnected mycelia cells through and around the non-nutrient discrete particles thereby bonding the discrete particles together to form a self-supporting composite material.

This embodiment of the process produces a self-supporting biocomposite material comprising a substrate of non-nutrient discrete particles; an amount of an antifungal protein (AFP1) native to Streptomyces tendae dispersed within the substrate; and a network of interconnected mycelia cells extending through and around the discrete particles and bonding the discrete particles together.

The antifungal protein (AFP1) dispersed within biocomposite material impacts fungicidal properties to the biocomposite material and, in particular, renders the biocomposite material resistant to Trichoderma as is the case with natamycin dispersed within the biocomposite material.

In each of the above described embodiments, the bacterial strain (i.e. microbial species) may be obtained from various sources. However, in accordance with the invention, the bacterial strain is obtained from the feedstock for making a biocomposite material. To this end, the invention provides a process of isolating a microbial species from a feedstock; genetically processing the microbial species and then returning the microbial species to the feedstock.

In accordance with the invention, this process comprises the steps of obtaining a feedstock including non-nutrient discrete particles, a nutrient material and at least one native microbial species; isolating the native microbial species from the feedstock; subjecting the isolated native microbial species to genetic processing to transform the native microbial species into a genetically engineered microbial species having predetermined characteristics; and thereafter returning the genetically engineered microbial species into the feedstock.

The native microbial species in the feedstock of interest would be one of Bacillus spp., Streptomyces alboniger, Streptomyces natalensis, and Streptomyces tendae and the characteristics of interest are one of producing a bio-film containing poly-gamma glutamic acid (PGA) in the feedstock, producing melanin in the feedstock, producing natamycin in the feedstock and producing an antifungal protein (AFP1) native to Streptomyces tendae in the feedstock.

Of note, B. subtilis has the ability to produce durable endospores which allows the B. subtilis to be used in co-inoculation. In this respect, the spore inoculum provides a robust precursor that can be prepared in advance to material cultivation, stored, transported, and more easily introduced into the feedstock during manufacturing particularly during infield deployment applications.

Fungal Processing

In accordance with the invention, the fungus for making a biocomposite material is genetically engineered to have predetermined characteristics.

In one embodiment, the filamentous fungus is genetically engineered to express carbonic anhydrase (CA) and the step of co-cultivating in the process of making a biocomposite material is performed in an environment without regulation of carbon dioxide (CO₂) through external inputs, such as, by using incubation chambers to regulate the carbon dioxide in the growth environment.

In another embodiment, the filamentous fungus is of the genus Trametes and is genetically engineered to overexpress chlamydospore production to increase the ability of the fungus to disperse through the growth substrate.

In another embodiment, the filamentous fungus is genetically engineered to overexpress a chitin deacetylase (DCA) gene to increase material strength in the formed self-supporting composite material. Overexpressing a chitin deacetylase gene in the fungal genome alters important structural components in the fungal cell wall i.e., chitin and chitosan. Modulating these ratios changes the mechanical properties of the cell wall and the final performance of the resultant biocomposite material when cultivated with these mutant strains.

In another embodiment, the filamentous fungus is of the genus Ganoderma and is genetically engineered to overexpress the production of hydrophobins to enhance the mycelium skin on the cells of the formed self-supporting composite material. Overexpressing hydrophobins (i.e. increasing the levels of hydrophobins) enhances the aesthetics of the resultant biocomposite material, produces a water-proofing skin encapsulating the material, which prevents water from entering the composite material, and resists swelling from humidity.

In another embodiment, the filamentous fungus is of the genus Ganoderma and is genetically engineered to overexpress the ortholog Ganoderma genes BGS1 and BGS2 that encode the two β-1,3-glucan synthases therein to increase glucans in the cells of the formed self-supporting composite material.

These and other objects of the invention will become more apparent from the following more detailed description.

DETAILED DESCRIPTION

The two current main production strains of filamentous fungus used in manufacturing processes to make a composite material according to U.S. Pat. No. 9,485,917, are members of the genus Ganoderma and Trametes, respectively.

Initially, two types of ATMT vectors were constructed, one type for use in Ganoderma, and another for use in Trametes. Each utilized the pOSCAR plasmid backbone developed for gene deletion in fungi. See Paz, Z., Garcia-Pedrajas, M. D., Andrews, D. L., Klosterman, S. J., Baeza-Montañez, L., Gold, S. E. (2011), One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi. Fungal Genet. Biol. 48(7): 677-84.

Each vector had a hygromycin-resistance cassette regulated by the glyceraldehyde-3-phosphate dehydrogenase (GPD) controlling sequences native to that particular fungus. GPD has been used extensively to drive the expression of selectable markers in filamentous fungi, and in particular mushrooms (Kim et al., 2015, Current technologies and related issues for mushroom transformation. Mycobiology, 43(1): 1-8).

Use was made of two Agrobacterium-based transformation protocols for filamentous fungi found in the literature. One procedure was adapted from a protocol described by Kemppainen and Pardo (2011, Transformation of the mycorrhizal fungus Laccaria bicolor by using Agrobacterium tumefaciens. Bioengineered Bugs 2:1, 38-44) for the mushroom Laccaria bicolor and another used by Michielse et al. (2008 Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nature Protocols 3(10): 1671-1678) for the ascomycete fungus Aspergillus awamori.

Both fungal mycelia and fungal protoplasts (cell wall-less derivatives of mycelia) were used as the target tissue in the initial transformation experiments. Binary vector (pOSCAR) recombinant DNA plasmids were cloned in E. coli, and then transformed into Agrobacterium tumefaciens strain AGL-1, which already contained the Ti plasmid. The phenolic compound acetosyringone (AS) was used during the pre-cultivation of A. tumefaciens and also during co-cultivation with the fungus. Three different ratios of bacterium to fungus were used during co-cultivation, as well as three different temperatures (20° C., 22.5° C., 25° C.). After 4 days of co-cultivation, mycelia were transferred to selection plates, containing hygromycin at 50 μM to select for fungal transformants, and cefotaxin at 200 μM to select against Agrobacterium.

Work Flow of Agrobacterium Tumefaciens Mediated Transformation (ATMT)

Membranes were seeded with fungal mycelium, then infected with A. tumefaciens (AGL-1) harboring the recombinant DNA plasmid. Membranes with co-cultured Mycelium/AGL-1 were then transferred onto drug selection agar to remove AGL-1 and select for putative transformants. The mycelium was then sub-cultured for further isolation and PCR screening.

Transformation efficiencies between 15-30% were achieved using the ATMT platform with fungal mycelium.

Bacterium Processing

Identification and assembling of plasmids, controlling sequences, and drug marker cassettes for four primary bacterium strains were conducted

Transformation protocols were also developed and optimized to allow for the efficient DNA transfer of these constructs (DNA sequences on the engineered plasmids) into each of our bacterium strains.

Engineering toolkits were developed for wild type isolates of Bacillus spp., Streptomyces alboniger, Streptomyces natalensis, and Streptomyces tendae. The “toolkits” involved the procedures for 1) cultivation in the lab i.e., temperatures, culture conditions 2) refined DNA transformation methods and 3) Gene controlling sequences used to engineer DNA plasmids which are then transformed into the bacteria.

Optimized transformation protocols for Bacillus spp. were based on inducing DNA uptake by nutrient starvation to increase the competency of recipient cells. These approaches were then tuned to overcome the more recalcitrant nature of non-domesticated strains through increased cell numbers and optimized competent cell preparations and media components.

Strong constitutive promoters were also identified, and used to drive expression in the overexpression plasmids. These technical achievements were then successfully used to engineer the Bacillus strains to produce melanin, PGA, antifungals, and confer drug resistance when co-cultivated with the fungus in agricultural feedstocks.

All three Streptomyces strains were cultivated at various temperatures to establish optimal growth conditions, in addition to best incubation conditions related to DNA conjugation protocols.

Bacterium Strain Engineering and Co-Cultivation

Using the toolkits for the bacterium community, these strains were engineered for enhanced material features by co-cultivation in feedstocks with the fungus.

One of the goals was to develop a novel microbial community that incorporates other soil microbes such as Streptomyces spp., and Bacillus spp. in farm waste inoculum. This more diversified community allowed the introduction of more complex, and novel properties into the resultant materials than were achieved using fungal species alone.

Co-Inoculating PGA Producing Bacillus Strains

Poly-gamma-glutamic acid (PGA) is a polypeptide, which consists of D-and L-glutamic acid units linked between amino and carboxyl groups.

Due to PGA's viscous, water soluble, and biodegradable properties, PGA has gained momentum in the fields of food science, agriculture, and biomedical devices. PGA is the primary constituent in the biofilm produced by some Bacillus subtilis strains.

The biofilm-producing B. subtilis strains were cohabited within our assembled microbiome.

Co-cultivating PGA producing Bacillus strains with the production fungus, leveraged the sticky viscus biofilm produced by the Bacillus to enhance the grown-in place bio-resin to significantly increase the flexure strength of the co-cultivated material by two-fold.

Co-Inoculating Melanin Producing Bacillus Strains

A melanin producing Bacillus strain was co-cultured with the fungus to produce a radiation hard material.

In this regard, a melanin expression pathway was engineered, then transformed into a Bacillus strain (ECO-ISO) isolated from a production feedstock.

The engineered Bacillus strain was co-cultivated with pre-myceliated feedstocks to produce a novel material with light and energy absorption properties. Bacillus was isolated from feedstocks, engineered to produce melanin, then introduced back into the community, and co-cultivated to add enhanced material properties.

Streptomyces spp., as a Co-Cultivation Chassis

In this particular case, the production fungus was co-inoculated with a strain of Streptomyces natalensis that provides substrates with fungicidal properties.

The goal in this case was to develop a cultivation paradigm that can utilize raw non-sterilized feedstocks. These biological controls could, in turn, reduce or eliminate the need for sterilization of raw agricultural substrates prior to inoculation and provide relief for sterile controls throughout the material growth cycle and manufacturing process.

Eliminating the need for sterilization provides significant energy savings during manufacture. Also, these biomaterials could be grown and produced outside of a manufacturing facility, thus reducing infrastructure, and enabling “in field” production using various low-quality agriculture substrates specific to the region.

Streptomyces natalensis naturally produces low levels of pimaricin, also known as natamycin, which is a fungicide with the ability to bind to sterols found in fungal cell membranes, thus making the cell wall permeable and lysing the cell. This fungicide has greater activity against Ascomycete contaminants, versus production fungi Ganoderma or Trametes. Natamycin's affinity for non-basidiomycetes species make this particular antifungal an attractive target for expression during cultivated manufacturing.

The overexpression of the pimM protein was engineered. This pimM protein has been characterized as a positive regulator for the natamycin biosynthesis gene cluster. See Anton, A., Santos-Aberturas, J., Mendes, M. V., Guerra, S. M., Martin, J. F., Aparicio, J. F. (2007). PimM, a PAS domain positive regulator of pimaricin biosynthesis in Streptomyces natalensis. Microbiology 153, 3174-3183.

By up-regulating the expression of pimM, the total yield of natamycin produced by our Streptomyces natalensis strains was increased. In this respect, natamycin production in wild type Streptomyces natalensis is within the effective range of inhibition (5 ug/mL) when cultured in vitro (liquid media). However, the production of natamycin must be increased to produce a more potent drug titer in co-cultivated feedstocks. Engineering a bacterium strain with increased natamycin potency helps to reduce the bacterial loading needed for substrate bioburden mitigation, thus further reducing process and material cost.

A pimM overexpression construct was designed and cloned using Streptomyces specific constitutive promoter (ermE) and a second expression construct was designed and cloned utilizing the native pimM promoter sequence.

The constructs were constructed using Gibson Assembly and subcloned into DH5a, and then transformed into a DNA donor non-methylation E. coli strain. A conjugation protocol was optimized to transform the DNA constructs into the unique Streptomyces strain.

Due to the complexities and diversification of Streptomyces species, there is no universal standard protocol that works efficiently with all Streptomyces spp. Accordingly, a spore harvesting technique (determined spores are best DNA recipients) was developed and optimized and a conjugation method was developed and optimized for non-domesticated Streptomyces strains.

The conjugation protocol was implemented to transform the pimM constructs from the E. coli donor strain into S. natalensis.

The efficacy of inhibitory properties was tested in feedstocks without the addition of the fungus. Feedstocks were loaded with Trichoderma spores, a common mold contaminate. Engineered Streptomyces was then co-cultured in these contaminated feedstocks, and the inhibitory effects were recorded. A strong linear inhibitory effect of Trichoderma in feedstocks was demonstrated as a function of natamycin expression.

A Bacillus strain was engineered to express an antifungal protein (AFP1) native to Streptomyces tendae. AFP1 is a more attractive protein for expression in a non-native host than natamycin based on its simple expression pathway. AFP1 is produced by the expression of a single protein (87 amino acids). This antifungal protein is of particular interest because of its resistance to degradation in harsh environments. AFP1 is stable over a pH range of 1.5-12, is highly resistant to digestion via peptidases, and can retain 50% of the antifungal activity after 60 min heat treatments of 70-100° C.

Bacillus AFP1 overexpression plasmids were designed by cloning our Streptomyces AFP1-Mature sequence into our Bacillus backbone p1664. The mature version of the AFP1 sequence has been truncated (42 AA cleavage) to eliminate the need for post-translational modifications. A strong constitutive promoter Pveg with an optimized ribosomal binding site was cloned in front of the AFP1 ORF to drive expression. The full expression sequence was cloned between a 5′ and 3′ flanking region for the thrC locus native to Bacillus spp. Taking advantage of homologous recombination, we integrated our expression sequence into the thrC gene of the Bacillus genome.

Once the plasmid was assembled using Gibson Assembly, the plasmid was transformed into 10-Beta E. coli cells for propagation. The plasmid was then isolated and verified by PCR, digestion, and sanger sequencing. The sequence verified plasmid was then transformed into two different Bacillus strains; Bacillus subtilis_168 (B.s_168), and Bacillus subtilis_KO7 (B.s_KO7). B.s_168 is a common lab strain that we have already been able to co-cultivate with our fungus in feedstocks.

Putative transformants were recovered by drug selection plating, and PCR verified.

To determine if our constructs were successfully expressing AFP1 in Bacillus, we performed Sq-RT-PCR to test the AFP1 transcription levels in our engineered strains. Both engineered strains had robust AFP1 transcription levels, and the non-engineered wild type strains had no detectable expression as expected.

To establish the effectiveness of Bacillus KO7_AFP1 as an antifungal agent when co-cultivated in our agricultural feedstocks, we performed experiments where we co-cultured both our Bacillus KO7_AFP1 strain (1×10⁸ bac/1 g dry feedstock) and Trichoderma spores in standard feedstock blends of Ecovative Design, LLC of Green Island, N.Y., without the addition of a production fungus. The exclusion of fungus was intentionally done to eliminate any possible community interactions between the native fungal defense mechanisms (i.e., the secretion of antimicrobial compounds) which could interfere with the interpretation of the Bacillus KO7_AFP1 inhibitory effects.

Three experimental sets were prepared for each experiment; 1) Trichoderma spores only, 2) Trichoderma spores co-cultured with the empty vector Bacillus strain KO7, and 3) Trichoderma spores co-cultured with Bacillus KO7_AFP1.

The Trichoderma spore load remained constant across sets, and was orders of magnitude higher than typical ambient contamination titers. Once feedstocks were inoculated, the material was allowed to incubate at standard incubation parameters of Ecovative Design, LLC.

Samples were visually inspected for Trichoderma sporulation over the course of ten days. We observed complete Trichoderma inhibition in the feedstocks through the first six days when co-cultured with Bacillus KO7_AFP1, after which point, Trichoderma began to sporulate throughout the co-cultured feedstocks at reduced rates when compared to the Trichoderma only control sets. This six day window of mold inhibition provides ample time to allow a co-cultivated fungus to fully colonize the feedstocks and out compete any bioburden molds that may reside within the feedstocks at inoculation.

In addition to determining the inhibitory properties of Bacillus KO7_AFP1 as it relates to bioburden (Trichoderma spp.), we needed to assess the potential for any significant inhibition of the production fungus when co-cultured in feedstocks with Bacillus KO7_AFP1.

Standard Ecovative feedstock materials (blends) were co-inoculated with production fungus (Ganoderma spp.) and cultured Bacillus KO7 AFP1 at 1×10⁸ bac/1 g dry feedstock. The material was cultivated using Ecovative's manufacturing guidelines, and grown into 2″×6″×6″ testing plaques. The co-cultivated material was then tested for mechanical properties; ASTM D1621-16. Material co-cultured with Bacillus KO7 (chassis control) and Bacillus KO7_APF1 (AFP1 expression) yielded no significant detrimental effects on compressive modulus (composite strength). The high inoculation titers of chassis bacteria did not reduce fungal colonization even in the presence AFP1 expression.

We successfully demonstrated the construction of a Bacillus KO7_AFP1 strain capable of expressing high titers of AFP1 protein. We further demonstrated the ability of our engineered strain to inhibit known bioburden molds (i.e., Trichoderma spp., and C. sitophila) isolated in the facilities and manufacturing process of Ecovative Design using plate based assays, and competition assays in our production feedstock blends.

Bacillus KO7 has been successfully co-cultured with the Ecovative production fungus on feedstocks without any negative effects on fungal colonization and final material mechanical properties while maintaining high viable bacterium survivability in the microbial community. Additionally, when AFP1 is expressed in situ by this new Bacillus chassis, we observed no detrimental effects on fungal growth and material performance.

Fungal Strain Engineering

A fungal network was engineered to bind the feedstock matrix.

Leveraging the co-cultivation platforms and fungal strain engineering provides for increased growth and enhanced proliferation of a mycelium network throughout the inoculated feedstocks, and increased resistance to competing microbes native to the feedstocks or contaminants encountered during scaled cultivation.

Several fungal strains were genetically engineered to increase performance in the bio-manufacturing process as described in U.S. Pat. No. 9,485,917 through the increased distribution of inoculum spores in feedstocks and the upregulation of carbonic anhydrase (CA) to help reduce environmental cultivation infrastructure.

The upregulation of cell surface hydrophobins, B-glucans, and chitosan was performed to increase fungal resin properties and enhance material performance i.e., strength, and water resistance.

Enhanced Manufacturing Process—nrg1 and efg1 Regulation (Chlamydospore)

Chlamydospores are produced by some fungi as a survival strategy when exposed to harsh environmental conditions (nutrient depletion, temperature variations). From a practical point of view, these spores enable production of an inoculum using Trametes sp. that is more resistant to contamination, with the increased ability to disperse throughout the substrate providing better acquisition of available resources.

The spore induction pathway was engineered by overexpressing a positive transcriptional regulator egf1, and knocking out a negative transcriptional regulator gene sequence nrg1. Both efg1 overexpression, and nrg1 knockout strains were made.

Using an alternative approach to upregulating chlamydospore production in Trametes by overexpression of a positive regulator, a plasmid was designed and cloned with the potential to knockout the ortholog of nrg1,characterized as a negative transcriptional regulator in the chlamydospore induction pathway.

The knockout strategy was designed around homologous recombination. A hygromycin drug cassette was cloned between a 5′ and 3′ flanking region of the nrg1 gene. This setup allowed recombination between the homologous flanking regions thus flipping the drug cassette into the nrg1 ORF effectively interrupting or knocking out the gene.

Microscopy was performed to characterize the nrg1_KO mutant. The nrg1_KO was grown on ME agar for three days, then sampled for characterization on bright field microscopy to evaluate asexual spore formation. Knocking out nrg1 significantly upregulated the production of asexual anthrospores. Anthrospores are asexual spores that form in monokaryon basidiomycete fungus. When compared, the relative numbers of anthrospores between the wild type and nrg1_KO showed a 42% increase in asexual spore formation in the mutant.

The growth rate of nrg1_KO was also measured and compared to the wild type. Both the nrg1_KO and wild type were inoculated onto the center of ME agar plates in triplicate, and incubated at 24° C. The total area of growth was measured over a period of five days. A significant increase was observed in the growth rate of the nrg1_KO when compared to the wild type. Here, a more robust, faster growing strain was observed from the deletion of nrg1.

The macro-morphology of the nrg1_KO is also distinctly different from the wild type. The wild type maintains uniform and tight leading-edge growth, while the mutant displays a more reaching non-uniform morphology. This phenotype is typically associated with a nutritional “searching” behavior.

Enhanced Material Performance-Chitin Deacetylase (CDA) Expression

Fungi cell walls consist in part of glycoproteins, hydrophobins, chitin, and chitosan. The ratio of all these constituents contributes to cell wall structure (i.e., mechanical properties, and permeability). Chitosan is derived through the deacetylation of nascent chitin by various chitin deacetylase (CDA) proteins which hydrolyze the acetamido group in the N-acetylglucosamine units of chitin, thus generating glucosamine units and acetic acid.

A chitin deacetylase overexpression plasmid was engineered and cloned.

The plasmid contained the CDA1 ORF cloned from Saccharomyces cerevisiae S288c, and the expression driven by the glyceraldehyde-3-phosphate dehydrogenase GPD promoter.

The S. cerevisiae CDA1 gene was the first chitin deacetylase gene identified, and its function has been characterized using both in vitro and in vivo models. Assembled constructs were transformed into a production fungus and verified by PCR.

For each of the two mutants used, the insertion of the CDA1 expression construct was positioned at different sites within the fungal genome. In fungal genomes, it is not uncommon for expression levels of the same genes to be different when expressed from different locations on the genome. Because of this, both transformants generated from the same plasmid were screened. Each strain was characterized for micro-morphology, growth rates, antifungal properties, and the mechanical strength of material generated by these strains.

Feedstocks were inoculated with each strain, and cultivated in bags and then in plastic molds (tools) to set the geometry for mechanical testing plaques. On visual observation, all strains colonized the feedstocks well and in similar fashion.

Materials cultivated from the engineered CDA strains were tested for their mechanical properties (compressive modulus) and compared to a wild type strain. Compressive modulus is a function of material stiffness and a standard metric used to assess material strength.

Material cultivated with the CDA1.18-3 strain, had significantly higher compressive modulus when compared to both wild type and CDA1.14-2. No significant differences were observed between CDA1.14-2 and the wild type.

Material cultivated with the CDA1.18-4 engineered strain was significantly stronger (compressive modulus) than material grown with the wild type strain.

Enhanced Material Performance—frt1 Regulation (Hydrophobins)

Hydrophobins are cysteine rich proteins that are anchored on the outer surface of fungal cell walls. These proteins give the outside surface of the cell its hydrophobic properties. Hydrophobins are unique to fungi, and are linked to growth morphology and cell signaling.

Two separate publicly available materials from Ecovative Design, LLC of Green Island, N.Y., were cultivated using unique cultivation paradigms and agricultural feedstock blends; 1) Ecovative's standard MycoComposite™ mycelium bound agricultural byproduct sold as “Protective Packaging” used as protective packaging or molded shapes, 2) Ecovative's MycoComposite™ 584 structural material used for construction building material.

Each of the two materials were cultivated and processed to either induce or reduce the amount of the mycelium skin that encapsulates the final forms. By reducing or inducing the amount of mycelium skin, we can quantitate the effects of the hydrophobin layer as a function of water absorption.

For standard MycoComposite™ Protective Packaging materials, environmental conditions such as temperature, CO2, and relative humidity (RH) were tuned to drive mycelial growth to the outer surface of the material. As such, Ecovative's structural materials were grown in conditions which enabled optimal internal colonization to add overall mechanical strength properties, thus reducing the external myceliation and reducing the hydrophobin skin on the surface of the material.

Once testing plaques were grown and processed to either reduce or induce the quantity of mycelium skin on the surface of the parts, they were subjected to water submersion testing (ASTM C1134). Parts were measured (physical dimensions and weight) and then submerged into a water tank. The plaques were held to complete submersion for 24 hours, then removed and re-measured. The percent of water mass absorbed was calculated and plotted for each of the two materials with variable mycelium skins. Water absorption of the standard Structural material was measured at 35% water mass absorption, but when the hydrophobic skin was reduced, there was a 61% water mass absorption for the material. This is a 43% increase in water absorption.

Standard MycoComposite™ Protective Packaging material was measured at a 15% water mass absorption, but significantly increased to 55% when the hydrophobic skin was removed demonstrating a staggering 77% increase in water absorption.

The amount of hydrophobic mycelium skin coating the part is proportional to the percent of water absorption. Both materials have similar absorption performance at reduced skin formats, but the MycoComposite™ Protective Packaging material performs better than the structural material in the induced hydrophobin skin sets. One explanation for this difference would be the paradigm which MycoComposite™ Protective Packaging is grown when compared to the Structural materials. MycoComposite™ Protective Packaging is cultivated to “force” myceliation to the outer surfaces of the material to aid in aesthetics (softer feel), and to promote “cushioning” properties gained with reduced internal colonization.

As for MycoComposite™ 584 structural materials, they are primarily cultivated to increase internal colonization with limited surface flush, thus enhancing mechanical properties such as internal bond and modulus. Engineering the fungus to increase hydrophobin production while still driving internal part colonization enables the growth of strong structural materials while retaining some of the critical mycelium hydrophobins on the surface of the material to protect the grown material from the elements.

Enhanced Material Performance—Regulation of β-Glucans

Glucans are the major structural polysaccharides of the fungal cell wall, constituting approximately 50-60% of the wall by dry weight. These polysaccharides are of particular interest with regards to increasing the internal bond strength of composites through enhanced fungal resin properties. The most abundant glucan in the fungal cell wall is β-1,3-glucan, which makes up between 65% and 90% of the whole β-glucan content.

Recombinant DNA constructs were made with the goal of over-expressing the genes (BGS1 and BGS2) that encode the two β-1,3-glucan synthases found in the Ganoderma genome. Use was made of the controlling sequences from the constitutively-expressed gene encoding glyceraldehyde-3-phosphate dehydrogenase (GPD) to drive expression of BGS1 and BGS2.

These constructs were used in classical PEG-mediated co-transformation experiments using a second plasmid containing a resistance gene to the fungicide carboxin. Three carboxin-resistant co-transformants were verified via PCR to have the integrated BGS overexpression constructs: two with BGS1 (BGS1-1, 1-7), and a third with BGS2 (BGS2-1). Assays for glucan content suggested a significant increase in the β-glucan fraction in each of the three co-transformants (i.e. 165%, 135%, and 147%, respectively).

The engineered Ganoderma_BGS strains had about a two-fold increase in β-1,3-glucan levels in the cell wall fractions when compared to the unmodified wild type Gandoerma.

The invention thus provides unique techniques for incorporating reprogrammed (genetically engineered) bacterial and fungal components in a process of producing myceliated material.

The invention also provides a process of cohabitating both bacterial and fungal species together in a substrate of discrete particles and a nutrient material to improve existing processes of producing myceliated material and produce a new class of composite materials. 

What is claimed is:
 1. A process of making a biocomposite material comprising the steps of forming a substrate of non-nutrient discrete particles and a nutrient material; adding a filamentous fungus to said substrate; adding a Bacillus subtilis strain characterized in producing a bio-film with poly-gamma-glutamic acid (PGA) to said substrate; co-cultivating said fungus and said Bacillus subtilis strain in said substrate and allowing said fungus to digest said nutrient material in said substrate over a period sufficient to grow hyphae and to allow said hyphae to form a network of interconnected mycelia cells through and around said non-nutrient discrete particles thereby bonding said discrete particles together to form a self-supporting composite material.
 2. A biocomposite material comprising a substrate of non-nutrient discrete particles; a bio-film containing poly-gamma-glutamic acid (PGA) dispersed within said substrate; and a network of interconnected mycelia cells extending through and around said discrete particles and bonding said discrete particles together.
 3. A process of making a biocomposite material comprising the steps of forming a substrate of non-nutrient discrete particles and a nutrient material; adding a filamentous fungus to said substrate; adding a Bacillus subtilis strain characterized in producing melanin to said substrate; co-cultivating said fungus and said Bacillus subtilis strain in said substrate and allowing said fungus to digest said nutrient material in said substrate over a period sufficient to grow hyphae and to allow said hyphae to form a network of interconnected mycelia cells through and around said non-nutrient discrete particles thereby bonding said discrete particles together to form a self-supporting composite material.
 4. A biocomposite material comprising a substrate of non-nutrient discrete particles; an amount of melanin dispersed within said substrate; and a network of interconnected mycelia cells extending through and around said discrete particles and bonding said discrete particles together, said biocomposite material being characterized in being radiation hard.
 5. A process of making a biocomposite material comprising the steps of forming a substrate of non-nutrient discrete particles and a nutrient material; adding a selected fungus to said substrate; adding Streptomyces natalensis characterized in producing natamycin to said substrate; co-cultivating said fungus and said Streptomyces natalensis in said substrate and allowing said fungus to digest said nutrient material in said substrate over a period sufficient to grow hyphae and to allow said hyphae to form a network of interconnected mycelia cells through and around said non-nutrient discrete particles thereby bonding said discrete particles together to form a self-supporting composite material while allowing said Streptomyces natalensis to produce natamycin as a fungicide in said self-supporting composite material.
 6. A biocomposite material comprising a substrate of non-nutrient discrete particles; an amount of natamycin dispersed within said substrate; and a network of interconnected mycelia cells extending through and around said discrete particles and bonding said discrete particles together, said biocomposite material being characterized in having fungicidal properties.
 7. A biocomposite material as set forth in claim 6 wherein said biocomposite material is resistant to Trichoderma.
 8. A process of making a biocomposite material comprising the steps of forming a substrate of non-nutrient discrete particles and a nutrient material; adding a filamentous fungus to said substrate; adding a Bacillus subtilis strain characterized in producing an antifungal protein (AFP1) native to Streptomyces tendae to said substrate; co-cultivating said fungus and said Bacillus subtilis strain in said substrate and allowing said fungus to digest said nutrient material in said substrate over a period sufficient to grow hyphae and to allow said hyphae to form a network of interconnected mycelia cells through and around said non-nutrient discrete particles thereby bonding said discrete particles together to form a self-supporting composite material.
 9. A biocomposite material comprising a substrate of non-nutrient discrete particles; an amount of an antifungal protein (AFP1) native to Streptomyces tendae dispersed within said substrate; and a network of interconnected mycelia cells extending through and around said discrete particles and bonding said discrete particles together, said biocomposite material being characterized in having fungicidal properties.
 10. A biocomposite material as set forth in claim 9 wherein said biocomposite material is resistant to Trichoderma.
 11. A process comprising the steps of obtaining a feedstock including non-nutrient discrete particles, a nutrient material and at least one native microbial species; isolating said at least one native microbial species from said feedstock; subjecting said isolated native microbial species to genetic processing to transform said native microbial species into a genetically engineered microbial species having predetermined characteristics; and thereafter returning said genetically engineered microbial species into said feedstock.
 12. A process as set forth in claim 11 wherein said native microbial species is one of Bacillus spp., Streptomyces alboniger, Streptomyces natalensis, and Streptomyces tendae.
 13. A process as set forth in claim 12 wherein said characteristics are one of producing a bio-film containing poly-gamma-glutamic acid (PGA) in said feedstock, producing melanin in said feedstock, producing natamycin in said feedstock and producing an antifungal protein (AFP1) native to Streptomyces tendae in said feedstock.
 14. A process of making a biocomposite material comprising the steps of forming a substrate of non-nutrient discrete particles and a nutrient material; adding a filamentous fungus genetically engineered to have predetermined characteristics to said substrate; adding a bacterial species to said substrate; and co-cultivating said fungus and said bacterial species in said substrate and allowing said fungus to digest said nutrient material in said substrate over a period sufficient to grow hyphae and to allow said hyphae to form a network of interconnected mycelia cells through and around said non-nutrient discrete particles thereby bonding said discrete particles together to form a self-supporting composite material.
 15. A process as set forth in claim 14 wherein said filamentous fungus is genetically engineered to express carbonic anhydrase (CA) and said step of co-cultivating is performed in an environment without regulation of carbon dioxide (CO₂) through external inputs.
 16. A process as set forth in claim 14 wherein said filamentous fungus is of the genus Trametes genetically engineered to overexpress chlamydospore production.
 17. A process as set forth in claim 14 wherein said filamentous fungus is genetically engineered to overexpress a chitin deacetylase (DCA) gene to increase material strength in the formed self-supporting composite material.
 18. A process as set forth in claim 14 wherein said filamentous fungus is genetically engineered to overexpress the production of hydrophobins to decrease water absorption of the formed self-supporting composite material.
 19. A process as set forth in claim 14 wherein said filamentous fungus is of the genus Ganoderma genetically engineered to overexpress the genes BGS1 and BGS2 that encode the two β-1,3-glucan synthases therein to increase glucans in said cells of the formed self-supporting composite material. 